There is an unfilled need for improved techniques to isolate and detect circulating tumor cells and other rare cells.
Most cancer-related mortalities result from metastasis. Cancer cells can be transported from the primary tumor by the circulatory system or bone marrow; some of these circulating cells may have metastatic potential. The ability to identify and count circulating tumor cells (CTCs) would be enormously helpful in the diagnosis and prognosis of many types of cancer. It has proven difficult, however, to reliably detect CTCs due to their extremely low concentration among a high background of “spectator” cells in peripheral blood (e.g., red and white blood cells). It has been reported that the concentration of CTCs in the blood correlates with mean survival time for breast cancer patients. It would be very useful clinically, for example, to be able to accurately count 0-10 CTCs in 1 mL of whole blood in a background of ˜109 erythrocytes and ˜106 leukocytes.
In sampling rare events from a large population, three important metrics are: (1) throughput, the number of cells identified or the number of sorting steps per unit time; (2) recovery, the fraction of the target cells successfully retrieved from the input sample; and (3) purity, the degree to which the recovered cells are free from “interfering” cells. In addition to these three metrics, the enriched cells must also be counted accurately.
Prior approaches to enriching CTCs in clinical samples have generally produced low recoveries with high purity, or low purity with high recovery. In a handful of cases, both high purity and high recovery have been reported, but only with highly specialized sample processing and handling equipment and techniques. For example, antibody-coated, micron-sized magnetic particles have been used to enrich CTCs with high purity, but only modest recoveries (˜70%). Polycarbonate membranes with varying pore sizes (8-14 μm) have been used to filter cells by size from relatively large volumes of blood (9.0-18 mL), with recovery of ˜85% of the CTCs, but at low purity due to the retention of large numbers of leukocytes as well.
Another approach has been to use quantitative PCR; or to use reverse-transcription PCR to assay mRNA as a surrogate for CTCs. RT-PCR can detect one CTC in an excess of 106 mononucleated cells. However, RT-PCR assays are prone to high inter-laboratory variability, are notoriously subject to false positives from environmental contamination, and require extensive sample handling and manipulation. Also, PCR techniques will generally destroy the cells being sampled.
Among the difficulties encountered by the existing methods for isolating and counting CTCs are one or more of the following: the need to select rare CTC cells from the mononucleated fraction of whole blood, which typically involves the use of density gradient centrifugation to remove the far more numerous RBCs; the need for flow cytometry apparatus; or the need for fluorescence microscopy. In addition to their cost and complexity, these procedures entail sample handling and transfer steps that can result in cell loss or contamination, which can dramatically affect results, particularly when one is dealing with a very low number of target cells to begin with.
Microfluidic systems can be used to process samples, including clinical samples, so as to minimize sample contamination and loss. However, microfluidic systems have not previously been widely used to process relatively large sample volumes (e.g., ≥1 mL) due to the small dimensions of the devices. For example, to fully process a 1.0 mL sample volume using a 30 μm×30 μm microchannel at a linear velocity of 1.0 mm s−1 would require ˜309 h (˜13 days). One approach has been to prepare a high-surface area immunological capture bed filled with microposts (e.g., ˜100 μm diameter×˜100 μm tall).
Patent application publication no. US2007/0026413A1 discloses a device with an array of obstacles (e.g., microposts) that relies on the hydrodynamics of flow through gaps between the obstacles, as well as spatial offsets between adjacent rows of the obstacles, and antibodies on the micropost surfaces to preferentially sort cell types by size, shape, chemical composition, or deformability. See also S. Nagrath et al., “Isolation of rare circulating tumour cells in cancer patients by microchip technology,” Nature, vol. 450, pp. 1235-1239 (2007); and patent application publication nos. US2007/0264675A1; US2007/0172903A1; US2008/0124721A1 and US2006/0134599A1.
Patent application publication no. US2008/0318324A1 discloses micro-fabricated or nano-fabricated devices for separating, concentrating, and isolating circulating tumor cells or other particles. Fluidic channels having particular cross-sectional shapes or other features could be used for dispersion, distribution, or partition of the fluidic flow, in order to reduce the direct impact of cells against exclusion features. The disclosure describes the use of so-called “effusive filtration”—redirecting, partitioning, dampening, or dispersing fluid flow to reduce physical impact on cells, while still allowing filtration through apertures. In some cases curvature might be included as a feature of the filtration perimeter. The channel surfaces could be treated with anticoagulant compounds, compounds that preferentially bind to circulating tumor cells, or compounds that prevent the sticking of cells.
P. Sethu et al., “Continuous flow microfluidic device for rapid erythrocyte lysis,” Anal. Chem., vol. 76, pp. 6247-6253 (2004) discloses a microfluidic device for erythrocyte removal from a blood sample by selectively lysing the erythrocytes. Whole blood was mixed with a lysis buffer, and the mixture was transported through a rectangular-wave microfluidic reaction channel.
M. Toner et al., “Blood-on-a-Chip,” Annu. Rev. Biomed. Eng., vol. 7, pp. 77-103 (2005) provides a review of research concerning the use of microdevices for manipulating blood and blood cells at the micro-scale.
Antibodies are not the only molecular recognition elements with high specificity for selected target molecules. Aptamers are another example of recognition elements that can have high specificity. Aptamers are single-stranded nucleic acid oligomers with specific affinity for a molecular target, generally via interactions other than classical Watson-Crick base pairing. In comparison to antibodies, aptamers (generally) have lower molecular weight and higher stability during long-term storage. Automated techniques are known in the art for selecting and synthesizing aptamers having specificity against a desired target, e.g., a membrane protein. Aptamers may readily be immobilized on solid support surfaces such as glass, polymers, or gold.
S. Lupold et al., Cancer Res., vol. 62, pp. 4029-4033 (2002) disclosed RNA aptamers directed against PSMA, and the use of those aptamers to target lymph node metastasis prostate cancer (LNCaP) cells.
J. Phillips et al., Anal. Chem., vol. 81, pp. 1033-1039 (2009) disclosed the use of aptamers on PDMS microchannel walls for the selection of leukemia cells seeded (˜1×106 cells/mL) in an aqueous buffer that was also loaded with non-cancerous cells.
A. Adams et al., “Low abundant biomarker screening in poly(methylmethacrylate) high aspect ratio microstructures using immunoaffinity-based molecular recognition,” Special Publication: Royal Society of Chemistry—Miniaturized Total Analysis Systems, vol. 1, pp. 132-134 (2004) disclosed a PMMA high aspect ratio, antibody-decorated, microfluidic device to pre-concentrate low abundant cancer cells from suspensions of simulated blood. An optimum flow rate for this device was found to be 2 mm/s. Antibodies were attached to a carboxylated polymer surface, generated by exposure to UV radiation. The device had 17 straight channels “with extreme rectangular character i.e. narrow (30-50 μm) and tall (250 μm)” to maximize collisions between cells and channel walls. A capture efficiency of 1% was reported for a 50 μm channel, and 100% for a 20 μm channel.